CN110776568B - Composite affinity column for purifying fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin - Google Patents

Composite affinity column for purifying fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin Download PDF

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CN110776568B
CN110776568B CN201911121740.XA CN201911121740A CN110776568B CN 110776568 B CN110776568 B CN 110776568B CN 201911121740 A CN201911121740 A CN 201911121740A CN 110776568 B CN110776568 B CN 110776568B
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toxin
zearalenone
monoclonal antibody
vomitoxin
toxins
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CN110776568A (en
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张奇
白艺珍
印南日
马飞
李培武
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Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
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Abstract

The invention relates to the purification of fumonisins B 1 Snake toxin, T-2 toxin, zearalenone, vomitoxin immunoadsorbent and composite affinity column. The immunoadsorbent comprises a solid phase carrier and fumonisin B coupled with the solid phase carrier 1 The monoclonal antibody is a monoclonal antibody secreted by hybridoma cell beads DAS5G11E7 with the preservation number of CCTCC NO: C201881. The affinity column prepared by the invention can be used for fumonisins B 1 The high performance liquid chromatography-mass spectrometry detection of the snake toxin, the T-2 toxin, the zearalenone and the vomitoxin has stable performance. Furthermore, the invention establishes an economic, rapid, accurate and safe detection method based on the affinity column, can be simultaneously used for the purification detection of the toxin samples, and has no mutual interference influence among the five types.

Description

Composite affinity column for purifying fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin
Technical Field
The invention relates to an immunoadsorbent for purifying fumonisin B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin and a composite affinity column.
Background
Fumonisins B1 are water-soluble metabolites produced by fusarium under certain temperature and humidity, and are diester compounds with similar structures and composed of different polyhydric alcohols and tricarballylic acid. Fumonisins B1 interfere with the normal physiological functions of plants in a low concentration range, are non-enzymatic compounds having toxic effects on plant metabolism, and belong to mycotoxins and non-host-specific toxins. Mainly distributed on crops mainly comprising corn, sorghum and wheat, and can cause agricultural economic losses such as seedling withering, root, stem and seed decay and the like. Fumonisins B1 can cause various specific toxicological effects on livestock, poultry and experimental animals, such as leukomalacia of horses and rabbits, which is manifested by symptoms of neurotoxicity, disturbance of consciousness, blindness, dyskinesia and the like, and even death of serious patients. Fumonisin B1 can also cause pulmonary edema and hydrothorax of pigs, liver and esophagus injuries, can also cause atherosclerosis of primates, liver cell apoptosis and nephrotoxicity of rats, lambs and calves, and has hepatotoxicity and carcinogenic effect, thereby bringing about serious economic loss to animal husbandry.
A snakelike toxin (DAS) is also called as sickle knife enol diacetate, is a metabolite of some strains of fusarium, belongs to a more important trichothecene compound in trichothecene compounds, and has higher pollution level and stronger acute toxicity of the trichothecene compounds with larger harm in the fusarium toxin. The serpentine toxin is colorless crystal, is difficult to dissolve in water, is soluble in polar solvent (such as methanol, etc.), is very stable, cannot be destroyed in the cooking process, has LDS of 0.75mg/kg for rats, and has strong thermal stability. The pollution level of the snake toxin in grains and feed is only slightly investigated in the United states, germany, italy and India, and the content of the snake toxin is 0.05-31.5 mg/kg. The snake toxin has strong toxicity, belongs to fat-soluble toxin, causes toxic symptoms similar to T-2 toxin, but is more serious, and has clinical manifestations of serious dermatitis, nausea, vomit, bloody diarrhea, bone marrow hematopoietic system damage, nervous system disorder anorexia and death after poisoning.
The T-2 toxin is the most toxic of the trichothecenes produced by Fusarium and it was discovered and reported by Bamburg et al in 1968. The fungi producing T-2 toxin mainly parasitize on the grains in the field, and most of the fungi belong to Fusarium, such as Fusarium sporotrichioides, fusarium pearium, fusarium trilobaculum and the like. The most suitable toxin-producing environment of the cladosporium is that the humidity is 40% -50%, and the temperature is 3-7 ℃; the most toxic producing ability in corn and rye is followed by barley, rice and wheat. Can cause poisoning of chicken, pig, rabbit, cat, rat, mouse, monkey, and pin. The toxicity is mainly manifested by cytotoxicity, skin toxicity, phytotoxicity, immunosuppression, emesia and the like. It has been discovered to date that T-2 toxin may be associated with four known human diseases, one of which is food toxic leukopenia (ATA); the other is bone disease-Kaschin-Beck disease (KBD), cartilage injury, etc.; thirdly, the reproductive development system is damaged; fourth, DNA damage of peripheral blood lymph cells.
Zearalenone, also known as the F-2 toxin, is first isolated from maize with gibberellic disease. Zearalenone is a strain of Fusarium such as Fusarium graminearum and Fusarium triloba. The zearalenone mainly pollutes corn, wheat, rice, barley, millet, oat and other grains, wherein the positive detection rate of the corn is 45 percent, and the highest toxic content can reach 2909mg/kg; the detection rate of wheat is 20%, and the poison content is 0.364-11.05 mg/kg. The zearalenone has strong heat resistance, and is completely destroyed after being treated at 110 ℃ for 1 hour. Zearalenone has estrogenic effects, and mainly acts on reproductive system, and can cause hyperestrogen in domestic animals, poultry and experimental mice. Various wheaten foods containing wheat flour with gibberellic disease can also cause poisoning symptoms of central nervous system, such as nausea, chills, headache, mental depression and ataxia.
Deoxynivalenol is a metabolite produced by nivalenol and fusarium avenae which often pollute wheat and parasitize grains. Deoxynivalenol, also known as vomitoxin (DON), is a secondary metabolite of fusarium. DON is mostly slowly produced in cereal crop seeds at low temperature, humidity and harvest seasons, mainly pollutes crops such as corns, wheat and the like, generally has higher concentration in wheat, barley, oats and corn seeds and lower concentration in rye, sorghum and rice, also pollutes grain products such as bread, biscuits, malted pointing melons and the like, and in addition, DON residues are found in milk and eggs of animals. DON is less toxic, but it is most likely to occur, and therefore has the highest incidence in agricultural products. The toxic effects of DON mainly affect the immune system and gastrointestinal tract of animals.
Currently, the mycotoxin detection methods mainly include thin layer chromatography, enzyme-linked immunosorbent assay (ELISA), immunoaffinity chromatography-liquid chromatography, immunoaffinity chromatography-fluorescence photometry, and the like. Thin layer chromatography requires contact with a large number of standards, is not good for experimenters' health, and has low sensitivity. The enzyme-linked immunosorbent assay is only suitable for qualitative detection, and the phenomena of false positive and false negative are easy to occur.
The immunoaffinity chromatography-liquid chromatography combines an immunoreaction with a chromatographic analysis method, utilizes the high specificity and affinity of antigen-antibody combination, combines a specific antibody to a chromatographic adsorbent by a chemical coupling bonding method, and realizes effective separation, enrichment and purification of target substances in a complex sample based on immunological reversible combination. Thereby, the mycotoxin in the sample can be specifically separated, and toxic solvents such as chloroform and dichloromethane are avoided. Therefore, the preparation of the purifying immunoaffinity column with stable performance is a prerequisite for establishing an economical, rapid, accurate and safe method for detecting the multi-toxin mixed pollution liquid chromatography of fumonisin B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an immunoadsorbent for purifying fumonisin B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin, a composite affinity column, and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
the immunoadsorbent for purifying fumonisin B1, serpentine toxin, T-2 toxin, zearalenone and vomitoxin is provided, the immunoadsorbent comprises a solid phase carrier and a fumonisin B1 monoclonal antibody, a serpentine toxin monoclonal antibody, a T-2 toxin monoclonal antibody, a zearalenone monoclonal antibody and a vomitoxin monoclonal antibody which are coupled with the solid phase carrier, and the serpentine toxin monoclonal antibody (anti-sickle enol diacetate monoclonal antibody) is a monoclonal antibody secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO: C201881. The hybridoma cell strain DAS5G11E7 is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 4 and 3, and the preservation address is China, wuhan and Wuhan university with the preservation number of CCTCC NO. C201881.
According to the scheme, the solid phase carrier is agarose gel.
Provides a composite affinity column loaded with immunoadsorbent of purifying fumonisin B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin antibody.
The preparation of the composite affinity column for purifying fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin comprises the following steps:
a) Substrate treatment
Washing CNBr-activated sepharose gel matrix powder with HCl under the condition of pH2-3 to remove impurities;
CNBr activated sepharose is provided in lyophilized form.
b) Ligand coupling
Dissolving fumonisin B1 monoclonal antibody, snakelike toxin monoclonal antibody, T-2 toxin monoclonal antibody, zearalenone monoclonal antibody and vomitoxin monoclonal antibody to be coupled by using a coupling buffer solution to obtain an antibody solution, and quickly transferring the activated sepharose gel matrix obtained in the step a) into the antibody solution for coupling;
c) Ligand blocking
Blocking all residual reactive groups;
d) Removing excess ligand that is not coupled after coupling;
e) And (5) column packing.
According to the scheme, the concentration of HCl used for washing in the step a) is 1mmol/L, and the washing time is 15min.
According to the above scheme, the coupling buffer in step b) is 0.2mol/L Na 2 HCO 3 ，pH8.3。
According to the above scheme, the concentration of each antibody solution in step b) is 10-15mg/mL.
According to the scheme, the coupling conditions of the step b) are as follows: the mixture is fully mixed for 2 to 4 hours at room temperature (20 to 25 ℃). According to the scheme, the ligand blocking process in the step c) comprises the following steps: transferring the agarose gel matrix treated by the step b) into 0.1mol/LTris-HCl buffer solution, and standing for 2-4h at room temperature.
According to the scheme, the step d) is as follows: washing the agarose gel matrix treated by the step c) by using buffer solutions with pH value of 4 and pH value of 8 in sequence for at least 3 cycles;
the buffer solution with pH value of 4 and pH value of 8 can be respectively 0.1mol/L acetic acid/sodium acetate buffer solution and 0.1mol/L Tris-HCl buffer solution.
According to the above protocol, step e) is carried out using 0.01% NaN based on 5 times the volume of the agarose gel 3 -PBS wash and use 0.01% NaN 3 PBS and then column packed.
According to the scheme, the IC50 of the anti-deoxynivalenol monoclonal antibody is preferably less than or equal to 15ppb; an anti-T-2 toxin monoclonal antibody having an IC50 of 2ppb or less; the fumonisin B1 antibody can be a monoclonal antibody secreted by a hybridoma cell strain Fm7A11 with the preservation number of CCTCC NO. C201636; the zearalenone antibody can be a monoclonal antibody secreted by a hybridoma cell strain 2D3 with the preservation number of CCTCC NO. C201328.
On the basis, the invention establishes a method for detecting contents of fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin by an immunoaffinity column purification-liquid chromatography-mass spectrometry, when a sample to be detected passes through the immunoaffinity column, an immunoadsorbent can specifically adsorb the fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin, other impurities flow out of the immunoaffinity column, then the affinity column is eluted by chromatographic grade methanol, the elution flow rate is 1 mL/min-2 mL/min, the fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin are eluted from the column, the sample is well purified, and the collected eluent is used for detection by a high performance liquid chromatography-mass spectrometer;
based on the method for detecting the contents of fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin by the composite affinity column, when a sample to be detected passes through the immunoaffinity column, an immunoadsorbent can specifically adsorb the fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin, other impurities flow out of the immunoaffinity column, then chromatographic grade methanol is used for eluting the affinity column, eluent is collected, namely the purified and concentrated sample is detected by a high performance liquid chromatography-mass spectrometer, and the content of each toxin is increased;
the conditions of the high performance liquid chromatography-mass spectrometer are as follows:
a mobile phase: a,0.05% formic acid/water solution; b,0.05% formic acid/acetonitrile solution;
b gradient elution: 0-3min,15% -50% B;4-5min,50% -70% B;6.5-8min,70% -100%; 8-10min, 100% -50% of (B); 10-11min,50% -15% by weight B;11-15min,15% by volume B.
c, chromatographic column: a C-18 column;
d, flow rate: 150-200 μ L/min;
e Mass Spectroscopy Scan parameters for various toxin detection are shown in Table 1
TABLE 1 scanning parameters for various toxins
Figure BDA0002275633050000051
The specific quantitative method can adopt the following modes: absorbing the fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin standard working solutions with different concentrations by using a sample injector, injecting the standard working solutions into a high performance liquid chromatography-mass spectrometer, measuring peak areas of the standard solutions under the conditions, drawing standard curves of various toxins, and then utilizing an external standard method to calculate the content of each toxin.
According to the scheme, the elution flow rate is 1 mL/min-2 mL/min.
The invention has the beneficial effects that: the affinity column prepared by the invention can be used for high performance liquid chromatography-mass spectrometry detection of fumonisins B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin, and has stable performance. Furthermore, the invention establishes an economic, rapid, accurate and safe detection method based on the affinity column, can be simultaneously used for the purification detection of the toxin samples, and has no mutual interference influence among the five types.
Drawings
Fig. 1 is affinity determination data of a sickle enol monoclonal antibody based on ribes diacetate provided by the present invention;
fig. 2 (a) shows the cross reaction result between the ribes diacetate fusarenol monoclonal antibody and other mycotoxins provided by the present invention; (b) The invention provides a standard curve of an enzyme linked immunosorbent assay method of ribeenol diacetate established by a ribeenol diacetate sickle cell monoclonal antibody.
Detailed Description
Obtaining of anti-diacetyl ribes fusarenol monoclonal antibody
The anti-diacetyl ribes fusarium enol monoclonal antibody is secreted and generated by a hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO. C201881, and the preparation method comprises the following steps:
injecting the hybridoma cell strain DAS5G11E7 into a BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, collecting ascites of the mouse, and purifying the antibody by adopting an octanoic acid-ammonium sulfate method, wherein the specific operation is as follows: filtering ascites of mice with double-layer filter paper, centrifuging at 4 deg.C and 12000r/min for more than 15min, sucking supernatant, mixing the obtained ascites supernatant with 4 times volume of acetate buffer solution, slowly adding n-octanoic acid under stirring, wherein the volume of n-octanoic acid required by each milliliter of ascites is 30-35 μ L, mixing at room temperature for 30-60min, and standing at 4 deg.C for more than 2 h. 12000r/min, centrifuging at 4 ℃ for more than 30min, discarding the precipitate, filtering the obtained supernatant by double-layer filter paper, adding phosphate buffer solution with the molar concentration of 0.1mol/L and the pH of 7.4 according to the volume of 1/10 filtrate, adjusting the pH of the mixed solution to 7.4 by using 2mol/L sodium hydroxide solution, slowly adding ammonium sulfate in an ice bath until the final concentration of the ammonium sulfate is 0.277g/mL, standing at 4 ℃ for more than 2h, then centrifuging at 12000r/min and at 4 ℃ for more than 30min, discarding the supernatant, resuspending the obtained precipitate by using phosphate buffer solution with the molar concentration of 0.01mol/L, pH of 7.4 according to the volume of 1/10 of the original ascites volume, filling the precipitate into a dialysis bag, dialyzing with 0.01mol/LPBS for two days, dialyzing again, taking out the protein solution in the dialysis bag for two days, centrifuging, collecting the supernatant, discarding the precipitate, pre-freezing the precipitate at-70 ℃ and freeze-drying in a freeze dryer. Collecting freeze-dried powder, namely the purified ribes diacetate resistant sickle enol monoclonal antibody;
the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water to 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to reach a constant volume of 100 mL; the 0.1mol/L phosphate buffer solution is prepared by adding water to 8g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium chloride and 0.2g of potassium dihydrogen phosphate to reach a constant volume of 100 mL.
The subtype of the ribes diacetate resisting sickle enol monoclonal antibody secreted by the hybridoma cell strain DAS5G11E7 is identified to be IgG2b by using a commercial subtype identification kit.
The titer of the antibody obtained by ascites purification of the mice can reach 3.2 multiplied by 10 measured by the conventional non-competitive enzyme-linked immunosorbent assay (ELISA) 5 I.e. 3.2X 10 dilution of antibody 5 The solution test result is positive. The sensitivity of the measurement on the sickle enol diacetate is 3.08ng/mL by using conventional indirect competition ELISA. The cross-reactivity with other mycotoxins, T2 toxin, HT2 toxin, vomitoxin, 3-acetyl deoxynivalenol, ochratoxin, fumonisin, was less than 0.01% (Table 1; FIG. 2). The specificity of the antibody can be evaluated by the cross-reactivity. Detecting DAS5G11E7 monoclonal antibody by indirect competition ELISA method, and detecting DAS, T2 toxin, HT2 toxin, DON, 3-ACDON, OTA, and FB 1 Preparing standard solutions with series concentrations, respectively adding the standard solutions and an antibody with the same volume into an ELISA plate, incubating for 1h, and performing other steps with an indirect competitive ELISA method. And (3) drawing a competitive inhibition curve by taking the concentration of the toxin standard substance as an abscissa and an OD value B/B0 measured by an enzyme-labeling instrument at 450nm as an ordinate, and judging the cross reaction rate by calculating the ratio of the IC50 values of the DAS and other toxins. The calculation formula is as follows:
CR% = (IC 50DAS/IC50 other toxins) × 100.
Table 1 cross-reaction of das5g1e7 with other toxins.
Figure BDA0002275633050000071
The affinity of DAS5G11E7 was determined using an indirect non-competitive ELISA. Coating an enzyme label plate with DAS-OVA according to the concentration of 1.0, 0.5, 0.25 and 0.125 mu g/mL, 100 mu L/hole, 37 ℃ and 2h; after blocking for 1h with the blocking solution, the antibody (dilution factor 1:2) diluted with PBS was added to the ELISA plate, and the rest steps were the same as the indirect non-competitive ELISA method. With the measured OD450 values as ordinate and the logarithm of the antibody concentration (mol/L) as abscissa, 4S-shaped curves of 4 concentrations were made. The maximum OD value at the top of each S-curve, i.e., ODmax, was found, and the antibody concentration corresponding to 50% ODmax of each curve was found. Any two of the 4 concentrations are combined in a group according to the formula Ka = (n-1)/2 (n [ Ab']t- [Ab]t) calculating the affinity constant of the antibody, wherein [ Ab']t、[Ab]t is the antibody concentration corresponding to the two 50% maximum OD values in each group, n is the fold of the envelope antigen concentration in each group (including three ratios of 1. The six obtained Ka values are averaged to obtain the anti-diacetyl-grass fusarenol mouse ascites antibody enzyme-linked immunosorbent assay (ELISA) method with the affinity of 5.4 multiplied by 10 8 L/moL (FIG. 1).
Screening of hybridoma cell strain DAS5G11E7
1. Animal immunization
A BALB/c mouse of 6-7 weeks old is immunized by using a sickle knife enol complete antigen DAS-BSA prepared in a laboratory. The first immunization is to inject the diacetyl grass fusarenol complete antigen and isovolumetric Freund's complete adjuvant into the subcutaneous multiple points of the back of the mouse neck after emulsification. The second immunization is carried out after 4 weeks, and the second immunization is emulsified by a Freund incomplete adjuvant and isovolumetric sickle enol complete antigen and injected into the abdominal cavity of a mouse. The third immunization was carried out at 4 weeks intervals from the second immunization in the same manner as the first immunization, and the fourth immunization was carried out 3 weeks after the third immunization in the same manner as the second immunization, which was also administered intraperitoneally. The 4 immunization doses were identical and were 70. Mu.g per mouse. And (3) collecting blood from tail veins 8-10 days after each immunization of the first 3 times, separating serum, and detecting the serum titer of the mice by adopting indirect ELISA. 8 days after 3 rd immunization, blood is collected by breaking the tail, and mice corresponding to serum with relatively high titer and sensitivity are selected for carrying out the last boosting immunization, wherein the immunization dose is 2 times of that of the mice.
2. Cell fusion
After 3 days of boosting immunity, adopting PEG with 50% of polyethylene glycol and molecular weight of 1450 as fusion agent, and making cell fusion according to conventional method, and its concrete steps are: killing the mouse by removing neck under aseptic condition, taking out spleen, grinding spleen with homogenizer, separating spleen cells with filter screen, mixing with mouse myeloma cell SP2/0 at a ratio of 5: 1, centrifuging, resuspending mixed cells with RPMI-1640 basic culture solution, centrifuging, and discarding supernatant. 50% of PEG was added thereto, 10 to 20mL of RPMI-1640 basic culture medium was added adherently for 1 minute at the time of use, centrifuged, the supernatant was discarded, the fused cells at the bottom of the tube were resuspended in 20mL of 1% HAT-containing cell complete medium, the suspended cells were added to 80mL of a semisolid medium, mixed well and added to a 6-well cell culture plate at 1.5 mL/well, and the mixture was cultured in a 37 ℃ carbon dioxide incubator. The complete cell culture medium containing 1% HAT contains 20% (volume percent) fetal bovine serum, 75% (volume percent) RPMI-1640 basic medium, 1% (weight percent) L-glutamine, 1% (volume percent) HEPES,1% (volume percent) diabody (10000 units per ml penicillin and 10000 micrograms per ml streptomycin), 2% (volume percent) growth factor (HFCS) and 1% (weight percent) hypoxanthine-aminopterine-thymidine, i.e., HAT and methylcellulose, which are purchased from sigma-Aldrich.
Screening and cloning of cell lines
And (3) picking out the clone from the culture medium by using a micropipette when the cell colony grows to be visible by naked eyes 2-3 weeks after the cells are fused, transferring the clone to a 96-hole cell culture plate, culturing by using HAT liquid, and sucking culture supernatant for detection when the cells grow to 2/3 of the hole bottom. Adopting a two-step screening method, wherein in the first step, an indirect ELISA method is adopted to screen out positive holes which resist sickle knife enol diacetate but not resist carrier protein BSA; the second step adopts indirect competition ELISA method to detect the positive holes screened in the first step, uses sickle enol diacetate as a competition source, and selectsSelecting the wells with higher absorbance and sensitivity (higher absorbance means higher final measurement value of the positive control well which is the well with 0 competitive antigen, and higher sensitivity means IC of the concentration of competitive antigen when the inhibition rate is 50% 50 Smaller value), carrying out subcloning by adopting a limiting dilution method, carrying out detection by adopting the same two-step method after subcloning, repeating the subcloning for 4-5 times, and obtaining a hybridoma cell strain DAS5G11E7. The hybridoma cell strain is preserved in China Center for Type Culture Collection (CCTCC) in 2018, 4 months and 3 days, wherein the preservation address is China, wuhan and Wuhan university, and the preservation number is CCTCC NO: C201881.
Determination of variable region sequence of anti-ribes diacetate fusarenol monoclonal antibody hybridoma cell strain DAS5G11E7 antibody
(1) Extracting total RNA: extracting total RNA capable of generating hybridoma cell strains DAS5G11E7 by adopting a total RNA extraction kit of Tiangen company according to an instruction;
(2) Synthesis of cDNA: taking the total RNA obtained in the step 1 as a template, taking oligo (dT) 15 as a primer and adopting SuperScript TM -2II reverse transcriptase instructions for reverse transcription to synthesize first strand cDNA; primer oligo (dT) 15 was purchased from Invitrogen;
(3) Cloning of variable region genes by PCR: designing primers according to conserved sites of mouse antibody gene sequences in GENBANK, and amplifying antibody heavy chain and light chain variable region genes by using CDNA as a template. The PCR procedure was: amplification is carried out for 30 cycles at 94 ℃ for 30s, at 58 ℃ for 45s, at 72 ℃ for 1min, and finally extension is carried out for 10min at 72 ℃. After the PCR product is separated by agarose gel electrophoresis of 1 percent (weight percentage), a kit is used for purifying and recovering a DNA fragment, the DNA fragment is connected in a vector pMD18-T, escherichia coli DH5 alpha competent cells are transformed, positive clones are picked up and sent to Suihai Sangni Biotechnology Co., ltd for sequencing. Wherein the sequences of the primers are respectively as follows: the heavy chain variable region primers were 5'-CAG GTS MAR CTG MAG GAG TCW G-3' (22 mer) and 5'-CAG GGG CCA GTG GAT AGA CAG ATG GGG G-3' (28 mer), where S, M, R and W are degenerate bases, M = a/C, R = a/G, S = G/C, W = a/T, and the light chain variable region primers were 5'-GAC ATC AAG ATG ACC CAG TCT CCA-3' (24 mer) and 5'-CCG TTT TAT TTC CAG CTT GGT CCC-3' (24 mer).
Results of the gene sequences obtained: the length of the gene sequence of the heavy chain variable region coding gene is 351bp, the sequence is shown as SEQ ID NO. 1, the heavy chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 117 amino acids, and the sequence is shown as SEQ ID NO. 3. The light chain variable region coding gene sequence is 324bp long and is shown as SEQ ID NO. 2, the light chain variable region coded by the gene sequence is deduced according to the obtained gene sequence and consists of 108 amino acids, and the sequence is shown as SEQ ID NO. 4.
Anti-fumonisins B 1 Obtaining of monoclonal antibody:
anti-fumonisins B 1 The monoclonal antibody is secreted and generated by a hybridoma cell strain Fm7A11 with the preservation number of CCTCC NO. C201636, and is prepared in advance according to a method reported in a patent with the application number of 2017101311660, and the preparation method comprises the following steps: injecting the hybridoma cell strain Fm7A11 into a BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, collecting ascites of the mouse, and purifying the antibody by adopting an octanoic acid-ammonium sulfate method, wherein the method comprises the following specific operations: filtering ascites of mice with double-layer filter paper, centrifuging at 4 deg.C and 12000r/min for more than 15min, sucking supernatant, mixing the obtained ascites supernatant with 4 times volume of acetate buffer solution, slowly adding n-octanoic acid under stirring, wherein the volume of n-octanoic acid required by each milliliter of ascites is 30-35 μ L, mixing at room temperature for 30-60min, and standing at 4 deg.C for more than 2 h. 12000r/min, centrifuging at 4 ℃ for more than 30min, discarding the precipitate, filtering the obtained supernatant by double-layer filter paper, adding phosphate buffer solution with the molar concentration of 0.1mol/L and the pH of 7.4 according to the volume of 1/10 filtrate, adjusting the pH of the mixed solution to 7.4 by using 2mol/L sodium hydroxide solution, slowly adding ammonium sulfate in an ice bath until the final concentration of the ammonium sulfate is 0.277g/mL, standing at 4 ℃ for more than 2h, then centrifuging at 12000r/min and at 4 ℃ for more than 30min, discarding the supernatant, resuspending the obtained precipitate by using phosphate buffer solution with the molar concentration of 0.01mol/L, pH of 7.4 according to the volume of 1/10 of the original ascites volume, filling the precipitate into a dialysis bag, dialyzing with 0.01mol/LPBS for two days, dialyzing again, taking out the protein solution in the dialysis bag for two days, centrifuging, collecting the supernatant, discarding the precipitate, pre-freezing the precipitate at-70 ℃ and freeze-drying in a freeze dryer. Collecting the freeze-dried powder to obtain the purified fumonisin B 1 A monoclonal antibody;
the acetate buffer solution is 0.29g of sodium acetate, and 0.141mL of acetic acid is obtained by adding water to a constant volume of 100 mL; the 0.01mol/L phosphate buffer solution is prepared by adding water into 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of potassium dihydrogen phosphate to reach a constant volume of 100 mL; the 0.1mol/L phosphate buffer solution is prepared by adding water to 8g of sodium chloride, 2.9g of disodium hydrogen phosphate dodecahydrate, 0.2g of potassium chloride and 0.2g of potassium dihydrogen phosphate to reach a constant volume of 100 mL.
Obtaining monoclonal antibody of zearalenone
The zearalenone monoclonal antibody is secreted and produced by a hybridoma cell strain 2D3 with the preservation number of CCTCC NO. C201328, and is specifically prepared in advance according to a method reported in a patent with the application number of 201310115825.3, and the preparation method comprises the following steps: injecting the hybridoma cell strain 2D3 into a BALB/c mouse which is treated by Freund's incomplete adjuvant in advance, collecting ascites of the mouse, and purifying the antibody by adopting an octanoic acid-ammonium sulfate method, wherein the method comprises the following specific operations: filtering ascites of a mouse by using double-layer filter paper, centrifuging for 15min at 4 ℃,12000r/min, sucking a supernatant, mixing the obtained ascites supernatant with 3 times of volume of acetate buffer solution, slowly adding caprylic acid under stirring, mixing for 30min at room temperature, standing for 2h at 4 ℃, then centrifuging for 30min at 12000r/min at 4 ℃, discarding a precipitate, filtering the obtained supernatant by using the double-layer filter paper, adding a phosphate buffer solution with the molar concentration of 0.1mol/L and the pH value of 7.4 in a 1/10 filtrate volume, adjusting the pH value of the mixed solution to 7.4,4 ℃ by using a 2mol/L sodium hydroxide solution, precooling, slowly adding ammonium sulfate until the final concentration of 0.277g/mL, standing for 2h at 4 ℃, centrifuging for 30min at 4 ℃,12000r/min, discarding the supernatant, suspending the obtained precipitate with a 0.01mol/L phosphate buffer solution with the original ascites volume of 1/10, loading the obtained precipitate into a dialysis bag, fully dialyzed pure water, placing the dialyzed protein in a refrigerator for later use, freezing and purifying the freeze-dried zearalenone antibody at 70 ℃, and obtaining a freeze-dried zearalenone antibody for later use;
the acetate buffer solution is obtained by adding 0.29g of sodium acetate and 0.141mL of acetic acid into water to fix the volume to 100 mL; the 0.1mol/L phosphate buffer solution is obtained by adding 0.8g of sodium chloride, 0.29g of disodium hydrogen phosphate dodecahydrate, 0.02g of potassium chloride and 0.02g of monopotassium phosphate into water to fix the volume to 100 mL.
The deoxynivalenol-resistant monoclonal antibody is preferably a deoxynivalenol-resistant monoclonal antibody with the IC50 less than or equal to 15 ppb. For example, the shandongdu biotechnology limited specifically used in this example is an anti-hypoxic nivalenol monoclonal antibody of shandongdu biotechnology limited, with an IC50 of 12ppb.
The anti-T-2 toxin monoclonal antibody is preferably an anti-T-2 toxin monoclonal antibody with an IC50 of 2ng/mL or less, such as Shandong DuoZhi Biotech, inc., which is specifically used in this example and has an IC50 value of 0.8ng/mL.
Example two:
preparation of fumonisin B1, snake toxin, T-2 toxin, zearalenone and vomitoxin composite immunoaffinity column
1. Preparation of the substrate
The desired 1g of CNBr-activated Sepharose lyophilized matrix powder (3.5 mL of swollen matrix formed per g of lyophilized matrix powder) was weighed out and dissolved in 1mmol/L HCl. The matrix will swell immediately and then be placed in a sintered glass filter and washed with 1mmol/L HCl for 15min.
2. Ligand (antibody) conjugation
a use of coupling buffer 0.2mol/L NaHCO 3 pH8.3 to be coupled with the fumonisin B1 antibody, snake toxin, T-2 toxin antibody, zearalenone antibody and vomitoxin antibody, the concentration of each antibody is 12.5 mg/mL, and the dissolved antibodies are placed in an ice bath for temporary storage. The above antibody-containing coupling buffer was added to a hermetically sealable container with a lid. CNBr activated Sepharose was quickly transferred to the antibody solution. The mixture is fully mixed for 2 to 4 hours at room temperature (20 to 25 ℃).
b calculation of coupling ratio: centrifugation at 2,000rpm, sepharose centrifugation to the bottom of the tubes, transfer of the supernatant to new centrifuge tubes and determination of the protein content value of the supernatant. The coupling ratio was calculated to be 98.5% (indicating that the coupling was successful). Sepharose centrifuged to the bottom of the tube was washed with a coupling buffer to remove excess ligand.
c, sealing: transfer the substrate to 0.1mol/L Tris-HCl buffer. Standing for 2-4h at room temperature, and blocking all residual active groups.
d for removing the excessive ligand which is not coupled after coupling, washing the substrate by using buffers with pH values of 4 and 8, namely 0.1mol/L acetic acid/sodium acetate buffer and 0.1mol/L Tris-HCl buffer, and washing for at least 3 cycles, wherein the usage amount of each buffer is at least 5 times of the volume of the substrate. Each washing cycle step: the washing was performed with 0.1mol/L acetic acid/sodium acetate buffer, followed by 0.1mol/L Tris-HCl buffer.
e 0.01% NaN with 5 gel volumes 3 PBS wash and use 0.01% NaN 3 PBS preservation.
3. The column was filled with a slurry prepared using a binding buffer and mixed at a ratio of 75% sedimentation matrix and 25% phosphate buffer (pH 7.0). The slurry was poured into the column in a continuous operation. The use of a glass rod that is inclined against the inner wall of the column to perform the column packing operation will help reduce the generation of bubbles. After the column is packed, the opening at the lower end of the affinity column is closed and the top member of the affinity column is removed. Carefully, PBS buffer, pH7.0, was added to the remainder of the packed affinity column to form an upward meniscus at the top of the column. The top frit was inserted into the affinity column at an angle to ensure that there was no air under the frit. Locking the sieve plate in place on the matrix surface, opening the opening below the affinity column, sterile filtering with 5 bed volumes of 0.01% 3 Column through PBS and use of 0.01% NaN 3 And (4) storing by PBS until the fumonisin B1, the snakelike toxin, the T-2 toxin, the zearalenone and the vomitoxin affinity column are filled and balanced, and directly using.
Example three: detection of fumonisin B1, S-toxin, T-2 toxin, zearalenone and vomitoxin in rice
1.0 detection of fumonisin B1, serpentine, T-2 toxin, zearalenone, vomitoxin in Rice
In the rice adding and recycling experiment, 500 mu g/kg,1000 mu g/kg and 2000 mu g/kg of fumonisins B1 and 10 mu g/kg,20 mu g/kg and 50 mu g/kg of snakes, T-2 toxins, zearalenone and vomitoxin are respectively added. Five parallel runs were made for each experiment.
Three gradients:
addition amount of the 1 st experiment: 500 mu g/kg fumonisin B1, 10 mu g/kg snake toxin, 10 mu g/kg T-2 toxin, 10 mu g/kg zearalenone and 10 mu g/kg vomitoxin.
Addition amount of experiment 2: 1000 mug/kg fumonisin B 1 20 mug/kg snake toxin, 20 mug/kg T-2 toxin, 20 mug/kg zearalenone and 20 mug/kg vomitoxin.
Addition amount of experiment 3: 2000. Mu.g/kg fumonisin B 1 50 mug/kg snake toxin, 50 mug/kg T-2, 50 mug/kg zearalenone and 50 mug/kg vomitoxin.
Extracting fumonisin B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin from rice:
accurately weighing 20.0g of the sample which is finely ground (the granularity is less than 2 mm), adding 100mL of acetonitrile/water/formic acid (80 +18+ 2), homogenizing, and extracting for 2min with high-speed stirring. Filtering with quantitative filter paper, accurately transferring 5.0mL of filtrate, adding 15.0mL of PH7.0PBS solution for dilution, and filtering with glass fiber filter paper for 1-2 times until the filtrate is clear. The composite immunoaffinity column was attached under a 10.0mL glass syringe. Accurately transferring 10.0mL of sample extracting solution into a glass syringe, connecting an air pressure pump with the glass syringe, and regulating the pressure to ensure that the solution slowly passes through the composite immunoaffinity column at the flow rate of about 6mL/min until 2-3 mL of air passes through the column. The column was rinsed 2 times with 10.0mL of water, all effluent discarded, and 2-3 mL of air was passed through the column. Accurately adding 1.0mL of chromatographic grade methanol for elution at the flow rate of 1 mL/min-2 mL/min, and collecting all the eluates in a glass test tube for detection.
2.0 high performance liquid chromatography-Mass Spectrometry conditions
a mobile phase: a,0.05% formic acid/water solution; b,0.05% formic acid/acetonitrile solution
b gradient elution: 0-3min,15% -50% B;4-5min,50% -70% B;6.5-8min,70% -100%; 8-10min, 100% -50% of (B); 10-11min,50% -15% by weight of B;11-15min,15% B.
c, chromatographic column: c-18 column (column length 50mm, inner diameter 2.1mm, packing diameter 1.7 μm)
d, flow rate: 200 mu L/min
e Mass Spectroscopy scanning parameters for the detection of various toxins are shown in Table 1
3.0 quantification of
Sucking fumonisins B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin standard working solution with different concentrations by using a sample injector, wherein the fumonisins B1 (5000, 2500, 1000, 200, 50, 5 and 1 mu g/kg); snake toxins (100, 50, 25, 10, 5, 1, 0.1 μ g/kg); t-2 toxin (100, 50, 25, 10, 5, 1, 0.1. Mu.g/kg); zearalenone (100, 50, 25, 10, 5, 1, 0.1 μ g/kg); vomitoxin (100, 50, 25, 10, 5, 1, 0.1 mug/kg) is injected into a high performance liquid chromatography-mass spectrometer, peak areas of standard solutions are measured under the conditions, standard curves of various toxins are drawn, and then the content of each toxin is calculated by an external standard method.
4.0 results
The standard recovery rate of the rice is between 82.5 and 109.1 percent, and the RSD is less than 10 percent. The result shows that the method completely meets the analysis requirements of detecting fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin in rice. The results are shown in tables 1 to 5, respectively.
TABLE 1 Fumonisin B in rice 1 Results of addition recovery
Figure BDA0002275633050000141
TABLE 2 Serpentine addition recovery results in rice
Figure BDA0002275633050000142
TABLE 3 recovery of T-2 toxin addition from rice
Figure BDA0002275633050000143
TABLE 4 zearalenone addition recovery results in rice
Figure BDA0002275633050000144
TABLE 5 results of vomitoxin addition recovery in rice
Figure BDA0002275633050000145
Example four: detection of fumonisins B1, snakes, T-2 toxins, zearalenone and vomitoxin in edible oil
1.0 detection of fumonisins B1, snakes, T-2 toxins, zearalenone and vomitoxin in edible oils
The standard addition recovery experiment of the edible oil is carried out, and 500 mug/kg, 1000 mug/kg and 2000 mug/kg fumonisins B with three concentration gradients are respectively added 1 And 10 mug/kg, 20 mug/kg, 50 mug/kg of three concentration gradients of snake toxin, T-2 toxin, zearalenone and vomitoxin. Five parallel runs were made for each experiment.
Extracting fumonisin B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin from edible oil:
extracting a vegetable oil liquid sample: accurately weighing 5.0g vegetable oil sample in a 50mL centrifuge tube, adding 15.0mL 70% methanol aqueous solution, mixing in a vortex mixer, shaking and mixing uniformly for 2min, centrifuging at 5 000r/min for 2min, transferring 10.0mL methanol solution layer, diluting with 20.0mL water, mixing uniformly in the mixer, filtering through glass fiber filter paper until the filtrate is clear. The composite immunoaffinity column was attached under a 10.0mL glass syringe. Accurately transferring 10.0mL of sample extracting solution into a glass syringe, connecting an air pressure pump with the glass syringe, and adjusting the pressure to ensure that the solution slowly passes through the composite immunoaffinity column at the flow rate of about 6mL/min until 2-3 mL of air passes through the column. The column was rinsed 2 times with 10.0mL of water, all effluent discarded, and 2-3 mL of air was passed through the column. Accurately adding 1.0mL of chromatographic grade methanol for elution at the flow rate of 1 mL/min-2 mL/min, and collecting all the eluates in a glass test tube for detection.
2.0 high performance liquid chromatography-Mass Spectrometry conditions
a mobile phase: a,0.05% formic acid/water solution; b,0.05% formic acid/acetonitrile solution
b gradient elution: 0-3min,15% -50% B;4-5min,50% -70% B;6.5-8min,70% -100% by weight B; 8-10min, 100% -50% of (B); 10-11min,50% -15% by weight B;11-15min,15% B.
c, chromatographic column: c-18 column (column length 50mm, inner diameter 2.1mm, packing diameter 1.7 μm)
d, flow rate: 200 mu L/min
e Mass Spectroscopy Scan parameters for various toxin detection are shown in Table 1
3.0 quantification of
Absorbing the fumonisins B1, snakelike toxins, T-2 toxins, zearalenone and vomitoxin standard working solutions with different concentrations by using a sample injector, injecting the standard working solutions into a high performance liquid chromatography-mass spectrometer, measuring peak areas of the standard solutions under the conditions, drawing standard curves of various toxins, and then utilizing an external standard method to calculate the content of each toxin.
4.0 results
The results of the addition recovery rates of the vegetable oil are all 88.5-109.2%, and the RSD is less than 10%. The result shows that the method completely meets the analysis requirements of detecting the rice fumonisins B1, the snakelike toxin, the T-2 toxin, the zearalenone and the vomitoxin. The results are shown in tables 6 to 10, respectively.
TABLE 6 Fumonisin B in vegetable oils 1 Results of addition recovery
Figure BDA0002275633050000161
TABLE 7 Serpentine addition recovery results in vegetable oils
Figure BDA0002275633050000162
TABLE 8 recovery of T-2 toxin addition in vegetable oils
Figure BDA0002275633050000163
TABLE 9 zearalenone addition recovery in vegetable oils results
Figure BDA0002275633050000164
TABLE 10 vomitoxin addition recovery results in vegetable oil
Figure BDA0002275633050000165
Figure BDA0002275633050000171
< 110 > institute of oil crops of Chinese academy of agricultural sciences
Less than 120, purifying fumonisin B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin composite affinity column
＜160＞ 4
＜210＞ 1
＜211＞ 351bp
＜212＞ DNA
< 213 > mice
＜400＞ 1
gaagtgcaac tggtggagtc tgggggagac ttagtgaagc ctggagggtc cctgaaactc 60
tcctgttcag cctccggatt cactttcaat tactatggca tgtcttgggt tcgccagact 120
ccagacaacc tcctggagtg ggtcgcaggc attagtagtg gtggttctta cacctattat 180
tctgacagtg tgaagggacg attcaccatc tccagagaca gtgccacgaa caccctgtac 240
ctgcaaatga ccagtctgaa gtctcaagac acagccatgt attattgtat tagactcccg 300
tttgggtcta tggactattg gggtcaagga accgcagtca ccgtctcctc a 351
＜210＞ 1
＜211＞ 324bp
＜212＞ DNA
< 213 > mice
＜400＞ 2
caggctgttg tgactcagga acctgcactc accacatcac ctggtgaaac agtcacactc 60
acttgtcgct caagtactgg ggctgtaaca actggtaatt atgtcaactg ggtccaagag 120
aaaccagatc atttattcag tggtctaata ggtaatacca ataaccgagc tccaggtgtt 180
cctgccagat tctcaggctc cctgattgga gacaaggctg ccctcaccat cacagggaca 240
cagactgagg atgaggcaat atatttctgt gctctatggt acaccgacca tttggtgttc 300
ggtggaggaa ccaaattgac tgtc 324
＜210＞ 1
＜211＞ 117
＜212＞ PRT
< 213 > mice
＜400＞ 3
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Asn Tyr Tyr
20 25 30
Gly Met Ser Trp Val Arg Gln Thr Pro Asp Asn Leu Leu Glu Trp Val
35 40 45
Ala Gly Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Thr Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Ser Gln Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ile Arg Leu Pro Phe Gly Ser Met Asp Tyr Trp Gly Gln Gly Thr Ala
100 105 110
Val Thr Val Ser Ser
115
＜210＞ 1
＜211＞ 108
＜212＞ PRT
< 213 > mice
＜400＞ 4
Gln Ala Val Val Thr Gln Glu Pro Ala Thr Thr Thr Ser Pro Gly Glu
1 5 10 15
Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Gly
20 25 30
Asn Tyr Val Asn Trp Val Gln Glu Lys Pro Asp His Leu Phe Ser Gly
35 40 45
Leu Ile Gly Asn Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Thr
65 70 75 80
Gln Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Thr Asp
85 90 95
His Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val
100 105

Claims (10)

1. Purify fumonisin B1, snakelike toxin, T-2 toxin, zearalenone, vomitoxin immunoadsorbent, its characterized in that: the immunoadsorbent comprises a solid phase carrier and fumonisin B1 monoclonal antibody, serpentine toxin monoclonal antibody, T-2 toxin monoclonal antibody, zearalenone monoclonal antibody and vomitoxin monoclonal antibody coupled to the solid phase carrier, wherein the serpentine toxin monoclonal antibody is generated by secretion of hybridoma cell strain DAS5G11E7 with the preservation number of CCTCC NO: C201881.
2. The immunoadsorbent of claim 1, wherein: the solid phase carrier is agarose gel.
3. The immunoadsorbent of claim 1, wherein: the IC50 of the anti-deoxynivalenol monoclonal antibody is less than or equal to 15ppb; an anti-T-2 toxin monoclonal antibody having an IC50 of 2ppb or less; the fumonisin B1 monoclonal antibody is selected from a monoclonal antibody secreted by a hybridoma cell strain Fm7A11 with the preservation number of CCTCC NO. C201636; the zearalenone monoclonal antibody is a monoclonal antibody secreted by a hybridoma cell strain 2D3 with the preservation number of CCTCC NO. C201328.
4. A complex affinity column for purified fumonisins B1, serpentine toxins, T-2 toxins, zearalenone, vomitoxin loaded with the immunoadsorbent of purified fumonisins B1, serpentine toxins, T-2 toxins, zearalenone, vomitoxin antibodies of claim 1.
5. The method for preparing the complex affinity column for purifying fumonisins B1, snakes, T-2 toxins, zearalenone, and vomitoxin according to claim 4, wherein the method comprises the following steps: the method comprises the following steps:
a) Substrate treatment
Washing CNBr-activated sepharose gel matrix powder with HCl under the condition of pH2-3 to remove impurities;
CNBr-activated sepharose is provided in lyophilized form;
b) Ligand coupling
Dissolving fumonisin B1 monoclonal antibody, snakelike toxin monoclonal antibody, T-2 toxin monoclonal antibody, zearalenone monoclonal antibody and vomitoxin monoclonal antibody to be coupled by using a coupling buffer solution to obtain an antibody solution, and quickly transferring the activated sepharose gel matrix obtained in the step a) into the antibody solution for coupling;
c) Ligand blocking
Blocking all residual reactive groups;
d) Removing excess ligand that is not coupled after coupling;
e) And (5) column packing.
6. The method for preparing the complex affinity column for purifying fumonisins B1, snakes, T-2 toxins, zearalenone and vomitoxin according to claim 5, wherein the method comprises the following steps: the concentration of HCl for washing in the step a) is 1mmol/L, and the washing time is 15min; the coupling buffer in step b) was 0.2mol/L Na 2 HCO 3 ，pH8.3。
7. The method for preparing a complex affinity column for purifying fumonisins B1, snakes, T-2 toxins, zearalenone and vomitoxin according to claim 5, wherein the affinity column comprises: the concentration of each antibody solution in the step b) is 10-15 mg/mL;
the coupling conditions of step b) are: mixing the mixture at room temperature of 20-25 deg.C for 2-4 hr.
8. The method for preparing a complex affinity column for purifying fumonisins B1, snakes, T-2 toxins, zearalenone and vomitoxin according to claim 5, wherein the affinity column comprises: the ligand sealing process of the step c) comprises the following steps: transferring the agarose gel matrix treated by the step b) into 0.1mol/L Tris-HCl buffer solution, and standing for 2-4h at room temperature.
9. The method for preparing the complex affinity column for purifying fumonisins B1, snakes, T-2 toxins, zearalenone and vomitoxin according to claim 5, wherein the method comprises the following steps: the step d) is: washing the agarose gel matrix treated by the step c) by using buffer solutions with pH value of 4 and pH value of 8 in sequence for at least 3 cycles;
respectively selecting 0.1mol/L acetic acid/sodium acetate buffer solution and 0.1mol/L Tris-HCl buffer solution from the buffer solutions with the pH value of 4 and the pH value of 8;
after the treatment in step d), 0.01% of NaN by 5 times the volume of the agarose gel 3 -PBS wash and use 0.01% NaN 3 PBS and then column packed.
10. The method for detecting the contents of fumonisins B1, snakes, T-2 toxins, zearalenone and vomitoxin based on the purified fumonisins B1, snakes, T-2 toxins, zearalenone and vomitoxin composite affinity column of claim 4, is characterized in that: when a sample to be detected passes through the composite affinity column for purifying fumonisin B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin according to claim 4, the immunoadsorbent can specifically adsorb fumonisin B1, snakelike toxin, T-2 toxin, zearalenone and vomitoxin, other impurities flow out of the immunoaffinity column, then the affinity column is eluted by chromatographic grade methanol, and the eluate, namely the purified and concentrated sample, is detected by a high performance liquid chromatography-mass spectrometer to obtain the content of each toxin.
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